Ozone-containing aqueous solution composition

Abstract

The present invention addresses mainly the problem of providing a novel composition or pharmaceutical that targets the mitochondrial network etc. and exhibits excellent anticancer activity while having a minimal effect on normal cells.　An example of the present invention is a composition comprising an aqueous solution that contains ozone and an activator (for example, a divalent iron salt, a flavin, or a nitrogen monoxide supplying agent), said composition being for inducing: fragmentation of mitochondria that have gathered uniformly in the vicinity of the cell nucleus of a cancer cell in a hypoxic state; accumulation of the fragmented mitochondria to one pole of the cell nucleus; and damage to the cell nucleus or death of the cancer cell after the accumulation.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of Japanese Application No. 2021-172840, filed with the Japan Patent Office on October 22, 2021. The entire application documents (specification, claims, drawings, abstract) are incorporated herein by reference for all purposes as if set forth herein. The present invention belongs to the technical field of anticancer drugs. The present invention relates to anticancer drugs that can selectively kill only cancer cells. The present invention also relates to ozone that can constitute such anticancer drugs. Solution It is related to. [Background technology]

[0002] Eukaryotic cells, including cancer cells, generally contain intracellular organelles called mitochondria, which are responsible for a variety of cellular functions, from energy production to macromolecular biosynthesis, oxidation-reduction (redox), and calcium ion homeostasis. Mitochondria play crucial roles in regulating cell proliferation, differentiation, and death, and are essential in the cell death signaling pathway. Mitochondria are highly flexible and motile organelles that change their shape, size, and localization in response to cellular conditions, such as energy demand, intracellular calcium, and reactive oxygen species (ROS) levels. The macroscopic structure of mitochondria (the mitochondrial network) is determined by the balance between fission and fusion, two opposing processes regulated by dynamin-related proteins (Drps) with guanosine triphosphate degrading activity. Because homeostasis of mitochondrial dynamics is essential for energy supply, metabolic activity, and mitochondrial DNA maintenance, disruption of mitochondrial dynamics leads to cellular dysfunction and death. Furthermore, mitochondria exhibit diverse intracellular distribution patterns, including a reticular network distributed throughout the cytoplasm (pan-cytoplasmic distribution) and division followed by uniform distribution near the nucleus (perinuclear mitochondrial clustering, PNMC). In recent years, it has become clear that the intracellular distribution of mitochondria is deeply involved in the plasticity, motility, invasiveness, survival, and death of cancer cells (Non-Patent Documents 1 and 2). Therefore, modulation of mitochondrial dynamics and intracellular distribution (hereinafter referred to as "kinetics") is an important target for the development of anticancer drugs as a potent means of inducing cancer cell death.

[0003] However, because control of mitochondrial dynamics is essential for the survival of not only cancer cells but also normal cells, indiscriminate deregulation may damage normal cells as well, potentially causing side effects. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] D. Pendin et al., Frontiers in Oncology, 2017 May 22; 7: 102. [Non-patent document 2] A. Altieri, British Journal of Cancer, 2017 Jul 25; 117(3): 301-305. Summary of the Invention [Problem to be solved by the invention]

[0005] The main objective of the present invention is to provide a novel composition or pharmaceutical product that targets mitochondrial dynamics and exerts excellent anti-cancer effects while having minimal effects on normal cells. [Means for solving the problem]

[0006] As a result of extensive research, the present inventors have found that ozone Dissolved Enhancers (also called adjuvants) )of The present inventors have found that by using a composition containing the compound, it is possible to selectively modulate mitochondrial dynamics in cancer cells, thereby selectively killing only cancer cells without affecting normal cells, and have completed the present invention.

[0007] The present invention includes, for example, the following. [1] An ozone solution characterized by dissolving ozone at a concentration of 2 to 5 mg / L as an active ingredient for inducing abnormal mitochondrial dynamics or cell death in cancer cells, and an activator. [2] The above-mentioned method, wherein the activator is one or more selected from the group consisting of a divalent iron salt, a flavin, a nitric oxide donor, and salinomycin and a pharmaceutically acceptable salt thereof. [1] Described Ozone dissolving liquid . [3] The above, wherein the divalent iron salt is ferrous sulfate, ferrous chloride, ferrous bromide, or ammonium iron(II) sulfate, the flavin is riboflavin, flavin mononucleotide (FMN), or flavin adenine dinucleotide (FAD), and the nitric oxide donor is organic nitrates, organic nitrites, metal nitrosyls, sydnonimines, S-nitrosothiols, or hydroxyimines. [2] Described in Ozone dissolving solution. [4] MammalContains mammalian cell culture medium or infusion preparations nothing, the above [1] Described Ozone dissolving liquid . [5] Oxygen-containing gas Ultraviolet light with a wavelength of 185 nm Ozone obtained by irradiating as the active species Contains do The gas is bubbled through an aqueous solution containing an activator. Ruka , or acid Soga vinegar of Ozone obtained by silent discharge as the active species Contains do gas But, It is not bubbled in an aqueous solution containing an activator. Ru, the above [1] Described in Ozone dissolving liquid .

[0008] [ 6 ] The method according to any one of [1] to [5] above. Ozone dissolving liquid Anti-cancer drugs, including [7] The above applies to cancers derived from epithelial cells, cancers derived from non-epithelial cells, leukemia, or lymphoma. [6] The anticancer agent according to any one of claims 1 to 4. [8] The above-mentioned cancer derived from epithelial cells is lung cancer, breast cancer, pancreatic cancer, colorectal cancer, gastric cancer, prostate cancer, ovarian cancer, oral cancer, or cancer derived from other organs, and the cancer derived from non-epithelial cells is osteosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, fibrosarcoma, liposarcoma, angiosarcoma, melanoma, neuroblastoma, or glioblastoma. [7] The anticancer agent according to any one of claims 1 to 4.

[0009] [9] Above [1]~ [5] any one of the above Ozone dissolving liquid Used to manufacture Decoration It is a position The apparatus has an ozone generating section for generating ozone, which is an activated gas, and a bubbling section for bubbling and dissolving the ozone generated in the ozone generating section in an aqueous solution containing an activator. Ozone generation characterized by Bubbling Device. [Effects of the Invention]

[0010] According to the present invention, Abnormal mitochondrial dynamics in cancer cellsThis can be induced to selectively kill cancer cells while leaving normal cells substantially unaffected. [Brief explanation of the drawings]

[0011] [Figure 1] 1 is a schematic diagram showing mitochondrial pan-cytoplasmic distribution, perinuclear clustering (PNMC), and monopolar mitochondrial perinuclear clustering (MPMC), and the effect of the composition of the present invention on cancer cells and normal cells. The morphology, intracellular distribution, and nuclear morphology of mitochondria at each stage are shown. [Figure 2] 1 is a conceptual diagram showing one embodiment of the configuration of an ozone generator according to the present invention. [Figure 3] FIG. 1 is a conceptual diagram showing another embodiment of the configuration of an ozone generator according to the present invention. [Figure 4] This figure shows the cell proliferation rate after administering pOBM (air-free ozone-containing gas produced by silent discharge bubbling into phenol red-free DMEM) to human oral squamous cell carcinoma SAS cells and culturing them for 72 hours. [Figure 5] FIG. 1 shows the cell proliferation rate after administering pOBM to human oral squamous cell carcinoma cells HOC-313 and culturing for 72 hours. [Figure 6] This figure shows the cell proliferation rate after 72 hours of culture in SAS treated with pOBM or NOR-3 aqueous solutions, either alone or in combination. The numbers in the figure represent the ozone concentration calculated from the dissolved ozone concentration of the original solution. [Figure 7] This figure shows the cell proliferation rate after 72 hours of culture in SAS treated with pOBM and riboflavin (vitamin B2, VB2) aqueous solutions, either alone or in combination. The numbers in the figure represent the ozone concentration calculated from the dissolved ozone concentration of the original solution. [Figure 8] FIG. 1 shows the cell proliferation rate after administering pOBM and an aqueous solution of salinomycin sodium, either alone or in combination, to HOC-313 cells and culturing them for 72 hours. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention will be described in detail below.

[0013] 1 The present invention Ozone dissolving liquid The present invention Ozone dissolving liquid (hereinafter referred to as "the composition of the present invention") is a composition comprising an aqueous solution containing ozone and an activator, Ozone is dissolved in an aqueous solution The present composition is characterized by its use in killing cancer cells. The composition can be used to fragment mitochondria uniformly clustered near the nucleus of hypoxic cancer cells, accumulate the fragmented mitochondria at a single pole on the nucleus, and induce nuclear damage or cancer cell death after accumulation. Therefore, the composition can induce hypoxic cancer cells to escape from the perinuclear clustering (PNMC) state and transition to a unipolar mitochondrial perinuclear clustering (MPMC) state, leading to nuclear damage or cell death. That is, the composition can function as a PNMC deregulator or MPMC inducer for hypoxic cancer cells. On the other hand, normal cells cannot survive hypoxic conditions and are not in the PNMC state under physiological conditions. Therefore, the composition is selective because it is believed to act only on cancer cells and not essentially affect normal cells.

[0014] 1.1 Induction of monopolar mitochondrial perinuclear clustering (MPMC) Cancer tissues are often hypoxic due to excessive cell proliferation and distance from blood vessels, resulting in insufficient oxygen supply. Hypoxic conditions trigger the hypoxic response through activation of hypoxia-inducible transcription factors (HIFs). Under hypoxic conditions, mitochondria diffusely present in the cytoplasm migrate to the perinuclear periphery and become uniformly localized at the nuclear periphery, a phenomenon known as nuclear periphery clustering (PNMC) (Figure 1A). PNMC is required for the formation of specific oxidized domains involved in binding to the dominant transcription factor (HIF-1α) and the hypoxia-responsive element (HRE), essential for the activation of hypoxia-inducible genes, thereby contributing to cancer cell survival. The composition of the present invention targets the intracellular deployment and localization of mitochondria. The composition of the present invention fragments mitochondria in cancer cells, causing the loss of PNMC (Figure 1B), and subsequently induces monopolar mitochondrial perinuclear clustering (MPMC), which aggregates mitochondria at a single pole on the cell nucleus (Figure 1C). The loss of PNMC and induction of MPMC leads to a loss of hypoxic adaptation, nuclear damage, and cell death. PNMC is not essential for normal cells that cannot survive under hypoxic conditions, but it is an intracellular phenomenon necessary for cancer cells to adapt to hypoxic conditions, and deregulation of PNMC results in a serious survival disorder only in cancer cells. Furthermore, MPMC is initiated by mitochondrial morphological changes triggered by superoxide production within mitochondria, and this superoxide production is selectively observed in cancer cells. In other words, the composition of the present invention can selectively damage or kill only cancer cells.

[0015] 1.2 Ingredients The composition of the present invention contains ozone and an activator. The activator is not particularly limited as long as it is a compound capable of promoting the cancer cell killing effect of ozone, or a compound capable of promoting the disappearance of PNMCs or the induction of MPMCs by ozone, and may also be a compound that functions as a reducing agent. Specifically, the composition of the present invention suitably contains an appropriate amount of one or more activators selected from the group consisting of, for example, divalent iron salts, flavins, nitric oxide donors, and salinomycin and its pharmaceutically acceptable salts. Furthermore, the composition of the present invention contains ozone and an activator. Dissolved The medium may consist of a mammalian cell culture medium or infusion fluid.

[0016] The concentration of dissolved ozone in the composition of the present invention is not particularly limited as long as the effects of the present invention are exhibited. mg / L ~5 mg / L It is appropriate that the range is within 0.3. mg / L ~3 mg / L Preferably, the range is 1 mg / L ~2 mg / L It is more preferable that the dissolved ozone concentration is within the range of 0.2 mg / L If the amount is less than 5, the effect of the present invention may not be exhibited. mg / L If the concentration is higher than this, it may affect normal cells. Dissolved ozone concentration can be measured by, for example, the 4-aminoantipyrine method, the iodine-starch reaction, or the ultraviolet method.

[0017] Examples of divalent iron salts include ferrous sulfate (FeSO), ferrous chloride (FeCl), ferrous bromide (FeBr), and ammonium iron(II) sulfate ((NH)Fe(SO). Among these, ammonium iron(II) sulfate is preferred. These may be used alone or in any combination of two or more.

[0018] Examples of flavins include riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD). Among these, riboflavin is preferred. These may be used alone or in any combination of two or more.

[0019] Examples of nitric oxide donors (NO donors) include nitrite ion, nitrate ion, organic nitrates, organic nitrites, metal nitrosyls, sydnonimines, S-nitrosothiols, and hydroxyimines. These may be used alone or in any combination of two or more.

[0020] Examples of such NO donors include NOR-1: (±)-(E)-4-methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexenamide, NOR-3: (±)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide, NOR-4: (±)-N-[(E)-4-ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexen-1-yl]-3-pyridinecarboxamide, and NOR-5: (±)-N-[(E)-4-ethyl-3-[(Z)-hydroxyimino]-6-methyl-5-nitro-3-heptenyl] Examples of suitable hydroxybenzoates include 1-hydroxy-2-oxo-3-(3-aminopropyl)-3-isopropyl-1-triazene, NOC-5: 1-hydroxy-2-oxo-3-(3-aminopropyl)-3-isopropyl-1-triazene, NOC-7: 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene, NOC-12: 1-hydroxy-2-oxo-3-(N-ethyl-2-aminoethyl)-3-ethyl-1-triazene, NOC-18: 1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene, and GSNO: S-nitrosoglutathione. Among these, NOR-3 is preferred. These may be used alone or in any combination of two or more.

[0021] Salinomycin is a polyether ionophore antibiotic with the chemical name (3R,5S,6S,7S)-3-[(2S,5S,7R,9S,10S,12R,15R)-2-[(2R,5R,6S)-5-Ethyl-5-hydroxy-6-methyltetrahydro-2H-pyran-2-yl]-15-hydroxy-2,10,12-trimethyl1,6,8-trioxadispiro[4.1.57.35]pentadec-13-en-9-yl]-6-hydroxy-7-[(2R,3S,6R)-6-[(R)-1-(hydroxy-12-methoxy)propyl]-3-methyltetrahydro-2Hpyran-2-yl]-5-methyloctan-4-one (CAS: No. 53003-10-4).

[0022] The composition of the present invention may contain salinomycin or a pharmaceutically acceptable salt thereof as an excipient. Salinomycin or a pharmaceutically acceptable salt thereof may be in the free form, a pharmaceutically acceptable salt thereof, a solvate such as a hydrate thereof, or an analog thereof. The pharmaceutically acceptable salt of salinomycin is not particularly limited as long as it is pharmaceutically acceptable and can form a salt of salinomycin, and examples thereof include base addition salts of salinomycin.

[0023] Examples of base addition salts include salts with inorganic bases and salts with organic bases. Examples of salts with inorganic bases include salts with sodium, potassium, magnesium, calcium, aluminum, etc. Examples of salts with organic bases include salts with methylamine, ethylamine, ethanolamine, lysine, ornithine, etc. Among these, the sodium salt of salinomycin is preferred.

[0024] The composition of the present invention may contain one or more components used in mammalian cell culture media, such as calcium chloride, potassium chloride, magnesium sulfate, amino acids such as arginine, cystine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, and valine (each of which preferably contains 10 to 600 mg / mL); and vitamins such as calcium pantothenate, sodium pantothenate, choline chloride, inositol, niacin, pyridoxal, riboflavin, and thiamine (each of which preferably contains 1 to 20 mg / mL).

[0025] The aqueous solution constituting the composition of the present invention can be a mammalian cell culture medium. Examples of such a medium include MEM (Eagle's Minimum Essential Medium), GMEM (Glasgow's Minimum Essential Medium), DMEM (Dulbecco's Modified Eagle's Medium), and IMDM (Iscove's Modified Dulbecco's Medium). Among these, DMEM is preferred, and DMEM without phenol red is more preferred. Phenol red may be, and preferably is not, contained in the composition of the present invention.

[0026] The aqueous solution constituting the composition of the present invention can be, for example, an electrolyte solution. Among these, infusion preparations for mammals are preferred, and infusion preparations for humans are even more preferred. Infusion preparations are frequently used to replenish fluids and electrolytes in humans and are therefore preferred from a safety perspective. Infusion preparations are not particularly limited as long as they are used in medical settings. Examples include hypotonic electrolyte solutions such as starter solution (No. 1 solution), dehydration replacement solution (No. 2 solution), maintenance solution (No. 3 solution), and postoperative recovery solution (No. 4 solution), as well as solutions containing glucose. Other examples include peripheral intravenous nutrition solutions containing glucose, electrolytes, amino acids, and water-soluble vitamin solutions, and hyperalimentation solutions containing glucose and electrolytes, or these in addition to amino acids, vitamins, trace elements, etc. Any commercially available solution can be used. Infusion solutions containing the above-mentioned activators or reducing agents can be used as is, while infusion solutions containing neither activators nor reducing agents can be used by adding an activator or reducing agent.

[0027] 1.3 Ozone bubbling liquid (OBM) In one embodiment, the composition of the present invention is oxidized in an oxygen-containing gas (e.g., air). Includes a spectrum with a wavelength of 185 nm ultraviolet Line The ozone-containing gas obtained by irradiation is bubbled in an aqueous solution containing the activator. Dissolved In another embodiment, the composition may be an acid. In pure gas to unvoiced Ozone-containing gas obtained by generating a discharge Su and bubbling in an aqueous solution containing the activator. Dissolved The bubbling can be carried out, for example, by inserting at least the tip of a nozzle into water or an aqueous solution and releasing an ozone-containing gas from the nozzle tip. 。

[0028] 2 Anticancer agent according to the present invention The anticancer agent according to the present invention (hereinafter referred to as "the anticancer agent of the present invention") contains the composition of the present invention. The ozone concentration in the anticancer agent of the present invention is not particularly limited as long as it is within a range that does not impair the effects of the present invention, and the appropriate and preferred amounts are the same as those in the case of the composition of the present invention.

[0029] The anticancer agent of the present invention can be widely applied to all cases that can be called cancer (malignant tumor), regardless of whether they are solid cancer or blood cancer. Examples of targets for application of the anticancer agent of the present invention include epithelial cell-derived cancer (carcinoma), non-epithelial cell-derived cancer (sarcoma), leukemia, and lymphoma. Examples of epithelial cell-derived cancer include lung cancer, breast cancer, pancreatic cancer, colon cancer, gastric cancer, prostate cancer, uterine cancer, ovarian cancer, and oral cancer. Examples of non-epithelial cell-derived cancer include osteosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, fibrosarcoma, liposarcoma, angiosarcoma, melanoma, neuroblastoma, and glioblastoma.

[0030] Some cancers (malignant tumors) activate various defense pathways that suppress apoptotic cell death, thereby exhibiting resistance to the cytocidal effects of anticancer drugs and radiation. Because the anticancer agent of the present invention can induce not only apoptosis but also multiple non-apoptotic cell deaths, it can also be used to treat intractable cancers that are resistant to existing multimodal therapies. Examples of such intractable cancers include osteosarcoma, melanoma, pancreatic cancer, and glioblastoma.

[0031] The anticancer agent of the present invention can be used in combination with other anticancer agents. Examples of such other anticancer agents include alkylating agents such as nitrogen mustards (e.g., cyclophosphamide, ifosfamide, melphalan, busulfan, thiotepa) and nitrosoureas (e.g., nimustine, ranimustine, dacarbazine, procarbazine, temozolomide, carmustine, streptozotocin, bendamustine), platinum compounds (e.g., cisplatin, carboplatin, oxaliplatin, nedaplatin), antimetabolites (e.g., 5-fluorouracil, cytarabine, gemcitabine, capecitabine, mercaptopurine, methotrexate, pemetrexed sodium), topoisomerase inhibitors (e.g., irinotecan, nogitecan, etoposide, zobuzoxacin), and microtubule inhibitors (e.g., vinblastine, vincristine, vindesine, vinorelbine, paclitaxel, docetaxel). inhibitors; antibiotics such as mitomycin C, doxorubicin, epirubicin, daunorubicin, bleomycin, pirarubicin, idarubicin, aclarubicin, amrubicin, and mitoxatrone; kinase inhibitors (e.g., gefitinib, erlotinib, osimertinib, afatinib, dacomitinib, imatinib, dasatinib, ponatinib, bosutinib, vandetanib, sunitinib, axinib, and ribozyme inhibitors); Examples of molecular targeted drugs include: tinib, pazopanib, lenvatinib, lapatinib, nintedanib, nilotinib, ibrutinib, gilitertinib, crizotinib, ceritinib, alectinib, and lorlatinib), mTOR inhibitors (e.g., everolimus and sirolimus), proteasome inhibitors (e.g., bortezomib, carfilzomib, and ixazomib), olaparib, niraparib, sorafenib, vemurafenib, dabrafenib, trametinib, palbociclib, and abemaciclib. Combination use of the anticancer agent of the present invention with existing anticancer agents can exert synergistic antitumor effects by enhancing apoptosis and inducing non-apoptotic cell death. As a result, reduced concentrations of anticancer agents with low tumor selectivity and reduced side effects are expected, making this an excellent complementary treatment.

[0032] The dosage form of the anticancer agent of the present invention is not particularly limited, and it can be formulated as, for example, a drip infusion, an injection, a spray, or an oral preparation. Among these, a drip infusion, an injection, or a spray is preferred.

[0033] 3. Ozone Generator According to the Present Invention The ozone generator according to the present invention (hereinafter referred to as "the apparatus of the present invention") is, in one embodiment, used to produce the composition of the present invention, as illustrated in FIG. , generating ozone as an activated gas species The generator comprises an air pump unit and an ozone generator unit, and is characterized in that air is introduced from outside the device using the air pump unit's intake function, and ultraviolet rays are irradiated by the ozone generator unit, and the air that has been irradiated with ultraviolet rays and has become an ozone-containing gas is released outside the device using the air pump unit's air supply function. The device of the present invention may also be equipped with an air tank for fluid pressure control (reducing pressure fluctuations, etc.). Furthermore, it may also be equipped with a pressure gauge, a flow meter, an air valve, a purge valve, etc., as necessary.

[0034] There are no particular limitations on the air pump unit as long as it functions as a gas pump having an air intake function and an air supply function. 。

[0035] There is no particular limitation on the pressure of the ozone-containing gas that can be released from the device of the present invention, but it is suitable to have a pressure in the range of 25 kPa to 50 kPa, for example. Among these, a pressure in the range of 30 kPa to 45 kPa is preferable, and a pressure in the range of 35 kPa to 40 kPa is even more preferable. 。

[0036] The device of the present invention comprises: To dissolve ozone The apparatus may be provided with an air bubbling section. If the ozone-containing gas delivered by the air pump section is released from the air bubbling section, the air bubbling section can be used for bubbling to produce the composition of the present invention. For example, an appropriate nozzle can be used for the air bubbling section.

[0037] The flow rate of the ozone-containing gas released from the air bubbling unit during bubbling is not particularly limited, but is suitably within the range of 0.1 L / min to 10 L / min, preferably within the range of 1 L / min to 8 L / min, and more preferably within the range of 2 L / min to 6 L / min.

[0038] The exhaust section of the device of the present invention can be equipped with an ozone scrubber that can remove ozone from the air flow before exhaust. By providing an ozone scrubber, the device of the present invention can perform highly safe exhaust. For example, manganese dioxide is widely used as a catalyst in ozone scrubbers, and known scrubber materials such as manganese dioxide can also be used in the device of the present invention. 。 [Example]

[0039] The present invention will be specifically explained below with reference to examples and test examples, but the present invention is not limited to the scope of the examples.

[0040] <Test Procedure> The test procedures common to each example are described below. 。

[0041] [ 1 ] Preparation and dilution of ozone aqueous solution The prepared ozone-containing gas was bubbled into phenol red-free DMEM (FR(-)DMEM) or purified water at a pressure of 36 kPa and a flow rate of 4 L / min to prepare an ozone aqueous solution. The dissolved ozone concentration was measured using the 4-aminoantipyrine method. Hereinafter, the prepared ozone aqueous solutions are referred to as OBM and pOBM, respectively, by bubbling AC ozone and AF ozone into FR(-)DMEM, and OBW, respectively, by bubbling AC ozone into purified water. These were then diluted with FR(-)DMEM or purified water.

[0042] [ 2 ] Measurement of cell proliferation rate APAM and ozone aqueous solutions were prepared according to the above test procedures [2] and [4], respectively. Non-water-soluble test drugs were dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 0.1% or less. Cells were suspended in DMEM containing 10% fetal calf serum (FCS) (FCS / DMEM) and seeded (5 × 10 cells) in a 96-well microplate. 4 The cells were incubated overnight in a CO2 incubator (95% air / 5% CO2 atmosphere), then treated with the test drug and incubated for an additional 72 hours. Cell proliferation was assessed using a water-soluble tetrazolium salt (WST-8) reduction method. This method utilizes the mitochondrial oxidoreductase activity of water-soluble tetrazolium salt to produce water-soluble formazan, and measures the absorbance at a wavelength of 540 nm to determine cell proliferation.

[0043] [ 3 ] Analysis of cell morphology and the morphology and subcellular localization of nuclei, tubulin, and mitochondria The cells were suspended in FCS / DMEM and seeded onto a polylysine-coated 35 mm dish (5 × 10 4 The cells were incubated overnight at 100°C (1000 cells / mL) and then treated with the test drugs for 18 hours. After washing, the cells were stained for nuclei, tubulin, and mitochondria with Hoechst 33342 (Hoe), Oregon Green Paclitaxel, and Mito Tracker Red (MTR), respectively, and their morphology was observed and photographed under a fluorescence microscope. Cell morphology was also observed and photographed simultaneously. As controls, cells were treated with non-plasma-irradiated DMEM or dimethyl sulfoxide (DMSO 0.1%) (which had no effect on any of the observed subjects).

[0044] [ 4 ]Caspase activation, membrane integrity, apoptosis measurement Seed (1 x 10) cells in a 6-well microplate. 5 The test drug was added to the cells (cells / mL), and after 24 hours of culture, APC-labeled annexin V and 7-amino-actinomycin D (7-AAD) (BD Biosciences) were added and the cells were analyzed by FACS Celesta. tmMeasurements were performed using a flow cytometer (BD Biosciences). Data were analyzed using Cell Quest Pro (Becton Dickinson Biosciences) and FlowJo software. Measurements were performed in triplicate, and annexin V-positive cells were defined as apoptotic cells.

[0045] [ 5 ]Analysis of intracellular ROS The cells were suspended in FCS / DMEM and seeded (5 × 10 4 After overnight incubation, the cells were treated with the test drug for 2 hours. After washing the cells, superoxide was released from the Mito SOX tm Hydrogen peroxide and hydroxyl radicals were detected by Hydrodrop Red (Thermo Fisher Scientific), respectively. tm and Oxi Orange tm Cells were stained with Fluorescence Intensity Imaging (Goryo Kayaku) and observed and photographed using an EVOS FL Cell Imaging System (Thermo Fisher Scientific). Data were analyzed using NIH ImageJ software (NIH, Bethesda, MD, USA).

[0046] [ 6 ] Protein analysis by Western blotting Cells were washed with calcium- and magnesium-free phosphate-buffered saline (PBS) and then lysed in Celllytic M Lysis buffer (Merck) containing a protease and protein phosphatase inhibitor cocktail (Sigma-Aldrich). The residual material was removed by centrifugation. The supernatant was collected, and protein was quantified using a BCA protein assay (Thermo Fisher Scientific). SDS-labeled proteins were separated by SDS-polyacrylamide gel electrophoresis using a 4-12% gradient gel (Invitrogen) and transferred to a PVDF membrane. The membrane was blocked with 0.2% Tween-20 in Tris-buffered saline (TBST) and 2% nonfat dry milk for 30 minutes, then incubated overnight at 4°C with primary antibodies in TBST containing 2% nonfat dry milk. After washing twice with TBST, the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hour at room temperature. The resulting membrane was washed three times with TBST, and the signal was detected using a chemiluminescence reagent (GE Healthcare) on an LAS-4000 (Fujifilm). 。

[0047] [Example 1] pOBM was tested according to the above test procedure [ 1 ] and used as the stock solution (100%). The dissolved ozone concentration was measured and diluted with FR(-)DMEM. The stock solution was prepared by bubbling ozone (AF ozone) into the solvent for 3 minutes per 10 mL to prepare an ozone solution. Cultured human squamous cell carcinoma cells SAS were administered pOBM (12.5-50%), and the cell proliferation rate after 72 hours of culture was measured according to the above test procedure [ 2 ] was measured according to (Fig. 4 ) In addition, cultured human squamous cell carcinoma cells HOC-313 were treated with pOBM (12.5-50%). Throw After 72 hours of culture, the cell proliferation rate was measured according to the above test procedure [ 2 ] was measured according to (Fig. 5 In both cases, control cells (Ctrl) were treated with FR(-) DMEM without ozone.

[0048] These results demonstrated that pOBM has potent anticancer activity. Furthermore, iron chelators suppressed the anticancer activity by approximately 50%, while catalase completely suppressed it, indicating that pOBM induces iron- and H2O2-mediated cytotoxicity.

[0049] [Example 2 ] pOBM was tested according to the above test procedure [ 1 ] and used as the stock solution (100%). The dissolved ozone concentration was measured and diluted with FR(-)DMEM. The stock solution was prepared by bubbling ozone (AF ozone) into the solvent for 1 minute per 10 mL to prepare an ozone solution. Human squamous cell carcinoma cells SAS were administered with pOBM (12.5%, 25%, 50%) and NOR-3 aqueous solution (NOR3 100 μM) alone or in combination, and after 72 hours of incubation, the cell proliferation rate was measured using the above test procedure [ 2 ] was measured according to (Fig. 6 In both cases, control cells (Ctrl) were treated with FR(-)DMEM without ozone. The NO donor NOR-3 showed synergistic antitumor effects when administered in combination with pOBM.

[0050] [Example 3 ] pOBM was tested according to the above test procedure [ 1 ] and used as the stock solution (100%). The dissolved ozone concentration was measured and diluted with FR(-)DMEM. The stock solution was made into an ozone solution by bubbling ozone (AF ozone) into the solvent for 1 minute per 10 mL. Human squamous cell carcinoma cells SAS were administered with pOBM (12.5%, 25%, 50%) and riboflavin (vitamin B2, VB2) aqueous solution (15 μM) alone or in combination, and after 72 hours of culture, the cell proliferation rate was measured using the above test procedure [ 2 ] was measured according to (Fig. 7 Riboflavin (vitamin B2), which is redox-active, showed synergistic antitumor effects when administered in combination with pOBM.

[0051] [Example 4 ] pOBM was tested according to the above test procedure [ 1] and used as the stock solution (100%). The dissolved ozone concentration was measured and diluted with FR(-)DMEM. The stock solution was made into an ozone solution by bubbling ozone (AF ozone) into the solvent for 3 minutes per 10 mL. Human squamous cell carcinoma cells HOC-313 were administered with pOBM (25%) and salinomycin sodium aqueous solution (salinomycin 2.5 μM) alone or in combination, and after 72 hours of culture, the cell proliferation rate was measured according to the above test procedure [ 2 ] was measured according to (Fig. 8 Salinomycin showed an adjuvant effect on pOBM. [Industrial Applicability]

[0052] The composition of the present invention can selectively kill cancer cells without substantially affecting normal cells. Furthermore, the device of the present invention can easily produce such a composition. Therefore, the present invention is useful in the medical industry, focusing on anticancer agents and their production devices.

Claims

1. An ozone solution characterized by dissolving ozone at a concentration of 2 to 5 mg / L as an active ingredient for inducing abnormal mitochondrial dynamics in cancer cells or cell death in cancer cells, and an activator.

2. 2. The ozone solution according to claim 1, wherein the activator is one or more selected from the group consisting of a divalent iron salt, a flavin, a nitric oxide donor, and salinomycin and a pharmaceutically acceptable salt thereof.

3. 3. The ozone dissolving solution according to claim 2, wherein the divalent iron salt is ferrous sulfate, ferrous chloride, ferrous bromide, or ammonium iron(II) sulfate, the flavin is riboflavin, flavin mononucleotide (FMN), or flavin adenine dinucleotide (FAD), and the nitric oxide donor is an organic nitrate ester, an organic nitrite ester, a metal nitrosyl ester, a sydnonimine, an S-nitrosothiol, or a hydroxyimine.

4. The ozone dissolving solution described in claim 1, which comprises a mammalian cell culture medium or an infusion preparation.

5. 2. The ozone solution according to claim 1, wherein the gas contains ozone as an active species obtained by irradiating an oxygen-containing gas with ultraviolet light having a spectrum with a wavelength of 185 nm, and the gas is bubbled in an aqueous solution containing an activator, or the gas contains ozone as an active species obtained by silently discharging oxygen gas, and the gas is bubbled in an aqueous solution containing an activator.

6. An anti-cancer agent comprising the ozone solution according to any one of claims 1 to 5.

7. The anticancer agent according to claim 6, which is applied to cancer derived from epithelial cells, cancer derived from non-epithelial cells, leukemia, or lymphoma.

8. The anticancer agent according to claim 7, wherein the cancer derived from epithelial cells is lung cancer, breast cancer, pancreatic cancer, colorectal cancer, gastric cancer, prostate cancer, ovarian cancer, oral cancer, or a cancer derived from another organ, and the cancer derived from non-epithelial cells is osteosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, fibrosarcoma, liposarcoma, angiosarcoma, melanoma, neuroblastoma, or glioblastoma.

9. 6. An ozone generating and bubbling device used to produce the ozone solution according to any one of claims 1 to 5, comprising: an ozone generating unit for generating ozone, which is an active gas; and a bubbling unit for bubbling the ozone generated in the ozone generating unit in an aqueous solution containing an activator, thereby dissolving the ozone.

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